SUMMARY Astrocytes are ubiquitous, highly branched cells that tile the entire central nervous system, making contacts with neurons and blood vessels, and serving diverse roles. Established roles include ion homeostasis, neurotransmitter clearance, synapse formation/removal, synaptic modulation and contributions to neurovascular coupling. From several of these perspectives, attention has focussed on astrocyte intracellular Ca2+ signaling as a basis to measure, interrogate and ultimately understand their roles within neural circuits. The focus on Ca2+ is based on knowledge that Ca2+ is a crucial second messenger and on the realisation that astrocytes are not electrically excitable. Thus, astrocyte Ca2+ signaling has been studied in vitro and in vivo for 25 years in a range of physiological settings. Based on these advances, however, a major current challenge is to reduce or otherwise abrogate (i.e. ?silence?) astrocyte calcium signaling and thus evaluate its functions in neural circuits. Such approaches are vital to advance basic brain research and they are of direct relevance to brain diseases in which astrocytes are implicated. As reported in the preliminary data of this application we developed a novel genetic strategy (called CalEx) to largely silence (but not abolish) astrocyte Ca2+ signaling in specific brain areas in vivo in adult mice. Using CalEx, we found compelling evidence for how astrocyte Ca2+ signaling regulates tonic GABAergic inhibition of striatal medium spiny neurons (MSNs) as well as striatum-dependent self- grooming behaviors in vivo. This renewal application will capitalise on these advances by combining state-of-the-art imaging and electrophysiology to test novel hypotheses and evaluate the functions of astrocyte Ca2+ signaling in adult striatal neural circuits in brain slices and in vivo. Aim 1 will evaluate how striatal astrocyte Ca2+ signaling affects neurons and astrocytes. In Aim 2, we will record MSN activity in brain slices and in vivo during silenced astrocyte Ca2+ signaling. Aim 3 will determine how silencing of astrocyte Ca2+ signaling alters gene expression and thus seek to identify molecular pathways that explain the hypothesis-driven experiments of Aims 1 and 2. All of the proposed aims are strongly supported by preliminary data. CalEx was extremely robust, and now permits exploration of a fundamental open question in neuroscience: what is the function of astrocyte Ca2+ signaling in circuits and in vivo? We will address this in a focussed way for the striatum and share our tools freely so others can use them in other brain areas.